Cells in healthy multicellular organisms are constantly subjected to mechanical processes that damage the cell membrane. This is especially the case for skeletal and cardiac muscle, and in many of the known muscular dystrophies sarcolemmal damage is exacerbated. The broad goal of the proposed studies is to define the molecular events that underlie repair of membrane damage, focusing on the recently discovered ability of one or more calpain proteases to facilitate membrane resealing and preserve viability in wounded cells. This discovery presents a new paradigm for intervention in membrane damage, and if the proposed studies are successful could lead to novel therapies to counteract muscle damage in muscular dystrophies. The first specific aim will focus on establishing the calpain enzyme(s) required to reseal damaged plasma membrane. By use of siRNA knockdown of calpains, we will establish which of these enzymes are required for repairing myoblasts and myotubes damaged by various established protocols, including mechanical cell injury, and exposure to laser pulses. We will focus on the ubiquitously distributed <- and m-calpains. Recently, we demonstrated that the blood plasma protein fetuin A stabilizes m-calpain and facilitates plasmalemma repair. The first part of specific aim 2 will entail investigation of fetuin A interactions with m-calpain that result in its increased ability to repair wounded plasma membrane. These studies have the potential to result in derivation of the first pharmacologic agent specifically designed to aid sarcolemma repair. In the second part of specific aim 2, we will investigate the role of talin in calpain-mediated sarcolemma repair. We have shown that talin, an important mediator of cytoskeletal and plasmalemma interactions, is cleaved by calpain immediately following scrape damage to fibroblast membranes. Talin also accumulated at injury sites of damaged muscle myotubes. By expression of non-cleavable mutant talin, talin head group, and talin knockdown with siRNA, we will explore two potential roles of talin proteolysis in membrane repair: 1) rapid dismantling of the actin cytoskeleton at the site of damage to allow regeneration of a new cortical cytoskeleton during repair;2) generation of talin head group to directly facilitate membrane reorganization during repair. Lastly, by confocal immunofluorescence microscopy and GST-fusion protein expression, we will explore the localization of calpains, activated calpains, dysferlin, talin, and annexins during sarcolemma repair, to gain information about how these putative mediators of membrane resealing may interact. PUBLIC HEALTH RELEVANCE. These studies are relevant to general public health because membrane damage occurs in many cell types in the body, and little is known about the mechanisms involved. Moreover, there is scientific evidence that some muscular dystrophies are associated with failure to repair ongoing muscle damage.